A bioinformatics tool for ensuring the backwards compatibility of Legionella pneumophila typing in the genomic era  M. Gordon, E. Yakunin, L. Valinsky, V. Chalifa-Caspi, J. Moran-Gilad  Clinical Microbiology and Infection  Volume 23, Issue 5, Pages 306-310 (May 2017) DOI: 10.1016/j.cmi.2017.01.002 Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 1 Mapping of reads to the mompS locus. The mompS locus usually harbours two gene copies. Amplification and Sanger sequencing using the sequence-based typing (SBT) scheme employs the non-specific forward primer 430F. Originally primers 492F and 1015R were used, but were abandoned because of their proximity to the allelic sequence and non-specificity, respectively. Per current ‘wet’ laboratory protocol, use of 430F and the reverse primer 1116R amplifies the ‘correct’ copy—mompS2 (687-bp amplicon in purple), so yielding consistent results, regardless of whether copies are identical or not. Primers 430F and 1015R are used as the Sanger sequencing primers (amber) and an internal 352-bp fragment is then analysed for typing (red). The proposed computational method initially aligns the reads to a unique reference sequence (spanning from new position 157 (red) to the start of 1116R) that is flanking and overlapping the mompS2 locus. Before the filtration process, all present paired reads are invariably aligned to the reference sequence. (a) Read alignment (grey) in the presence of identical mompS copies; (b) read alignment in the presence of non-identical copies (grey reads correspond to the ‘correct’ copy and white reads to the ‘wrong’ copy). Aligned reads cannot be allocated to their original gene copy. Clinical Microbiology and Infection 2017 23, 306-310DOI: (10.1016/j.cmi.2017.01.002) Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions

Fig. 2 mompS calling through selection of mompS2 unique reads. Mapped mompS reads are subject to variant calling. In the absence of single-nucleotide polymorphism (SNP) variation (‘homozygosity’), mapped reads belonging to either of the identical mompS1 and mompS2 alleles are called (not shown). In the presence of SNP variation (‘heterozygosity’), reads (grey and white, corresponding to the ‘correct’ and ‘wrong’ gene copies) are filtered. Different SNPs are shown as colour strips. Only paired-end reads overlapping at one end with the unique flanking ‘anchors’ (yellow areas) are retained through filtration, thereby ensuring only reads originating from the mompS2 locus (grey) are analysed. Clinical Microbiology and Infection 2017 23, 306-310DOI: (10.1016/j.cmi.2017.01.002) Copyright © 2017 European Society of Clinical Microbiology and Infectious Diseases Terms and Conditions